Section I. Biomolecule Separation

1. Two-Dimensional Electrophoresis in a Chip

Z. Hugh Fan, Champak Das and Hong Chen

Two-dimensional (2D) electrophoresis chips are fabricated from cyclic olefin copolymer resins using microfabrication and compression molding techniques. Protein separation in the chips is carried out by integrating isoelectric focusing (IEF) and polyacrylamide gel electrophoresis (PAGE). Each chip consists of one IEF channel for the first dimension and 29 parallel PAGE channels for the second dimension. The IEF and PAGE channels are intersected orthogonally so that the focused protein can be transferred from the first to the second dimension. An array of microfluidic pseudo-valves is created for introducing different separation media, without cross-contamination, in both dimensions. Fabrication of the valves is achieved by photo-initiated, in situ gel polymerization; acrylamide monomers are polymerized only in the PAGE channels whereas polymerization does not take place in the IEF channel where a mask is placed to block light exposure. A layer of titanium dioxide membrane can also be synthesized at the interface of two dimensions for strengthening the pseudo-valves. The presence of the valves does not affect the performance of IEF or PAGE when they are investigated separately. Detection in the chip is achieved using a laser induced fluorescence imaging system. Fluorescently-labeled proteins with either similar pI values or close molecular weight are well separated, demonstrating the potential of the 2D electrophoresis chips.

2. Liquid Chromatography in Microfluidic Chips

Hernan V. Fuentes and Adam T. Woolley

In this chapter, we provide a brief review of the "state of the art" of miniaturized devices for liquid chromatography. Instructions are included for the design, manufacture and application of glass microfluidic chips with integrated micropumps and microchannels for pressure-driven separations. Electrolysis-based micropumps with embedded electrodes were connected fluidically to sample or mobile phase reservoirs to effect sample injection and separation. We developed an on-chip pressure-balanced injection method, which allowed picoliter-range samples to be introduced into a microchannel with no dead volume. Microchannel walls were coated, resulting in a reversed-phase microfabricated open tubular column. The system was used to separate three fluorescently labeled amino acids in less than 40 s with good efficiency (3350 theoretical plates). We review recent efforts aimed at developing microfluidic systems for on-chip liquid chromatography and compare the advantages and disadvantages of our approach to those of others. On chip pressure-driven separations hold great potential to revolutionize many assays in which minute sample volumes must be analyzed fast and in parallel. Moreover, microchips with nL/min flow rates are easy to interface with mass spectrometry, eliminating many of the challenges of coupling conventional columns to electrospray sources.

3. Design and Fabrication of Microfluidic Devices for Flow-based Separation of Blood Cells

Lance L. Munn and Abhishek Jain

Enrichment of specific cell populations such as leukocytes and circulating cancer cells from a sample of whole blood is the required first step of many clinical and basic research assays. We are developing microfluidic devices that take advantage of the intrinsic features of blood flow in the microcirculation to separate cells directly from whole blood. These devices consist of simple networks of rectangular microchannels manufactured using soft lithography, a technology that allows rapid development of robust, but relatively complex devices capable of accommodating the flow of even dense solutions of blood cells. Polydimethylsiloxane (PDMS) molding is ideally suited for live cell separations because of the ability to coat the channels with various biologically-relevant molecules and the fact that the sizes of blood cells lie in the range of channel sizes easily produced in PDMS. Here we detail our motivation and methodology for producing separation devices using PDMS molding.

4. Hydrophoretic Method for Continuous Blood Cell Separation

Sungyoung Choi and Je-Kyun Park

Precise and rapid isolation of blood cells is of fundamental importance in clinical and biomedical researches. The preparation of white blood cells for downstream analysis is commonly accomplished by selective lysis of red blood cells and differential centrifugation, which are labor-intensive and require a large volume of blood samples. Recent technical advances of microfluidic devices for blood cell separation provide new capabilities for accurate and fast separation of a small number of blood cells. In the microfluidic environment, separation devices take advantage of accurate cell control without turbulent disturbance due to laminar flow at low Reynolds number and high process efficiency even with a small number of cells. This chapter introduces a microfluidic technology to separate blood cells by hydrophoresis, describing details of the separation principle and the individual steps necessary to perform the hydrophoretic separation. We simultaneously present a review of other methods for separation of blood cells, comparing their advantages and disadvantages. Finally, we present some challenges and future trends of microfluidic technologies for blood sample preparation.

5. Microchip Gel Electrophoresis of DNA with Integrated Whole-column Detection

Roger C. Lo and Victor M. Ugaz

Gel electrophoresis is an essential analytical step in a wide spectrum of DNA analysis assays. This importance has motivated efforts aimed at developing advanced microfluidic "lab-on-a-chip" systems that incorporate embedded electrophoresis capabilities. In this chapter, we describe recent work aimed at constructing a new automated whole-gel scanning detection system that allows microchip-based gel electrophoresis of DNA to be performed in a rapid, low-field, miniaturized format. This system is based on a versatile microfluidic platform that incorporates integrated on-chip electrodes, heaters, and temperature sensors coupled with instrumentation configured to enable the progress of a DNA separation to be continuously monitored along the entire microchannel in near real time. This is in contrast to both conventional slab gel imaging where the entire gel can be viewed but only at one point in time after completion of the separation, and capillary electrophoresis systems that permit detection as a function of time but only at a single downstream location.

6. Microscale Blood Separation Technology

Jeffrey D. Zahn, Sung Yang, Akif Undar and Pantelis Athanasiou

The purpose of microscale blood separation devices is to either identify individual cell types of interest within a mixed cellular population, concentrate (enrich) a single cell type or sort a mixed population of whole blood into subpopulations of similar cell types for downstream processing and analysis within biomedical microdevices. Conventional laboratory blood analyses serving these roles require well trained, skilled personnel using expensive and sophisticated equipment. The development of autonomous microdevices for blood cell separations is an enabling technology which allows rapid, reproducible laboratory grade tests for applications in point of care diagnostics, continuous patient monitoring with feedback controlled drug delivery and technologies to provide medical diagnostics and treatment. Blood cell diagnostics are utilized to diagnose a multitude of pathological conditions by monitoring changes in physiological blood plasma chemistry, inflammatory responses characterized by complement, neutrophil, and platelet activation, and subsequent release of pro-inflammatory cytokines, changes in blood cell populations, or identifying cluster of differentiation (CD) cell surface protein antigens used in the diagnosis of cancers and infection. Methods for cell separations exploit specific physical property differences between differing cell types which include: fluorescence-based, magnetic-based, affinity-based, electrical or dielectric property-based, cell size and density gradient-based separations depending on the properties of the cells of interest. Since these microdevices are very sophisticated, they should be designed to allow easy operation without technical training while providing advantages such as lower analysis cost or time to analysis over conventional laboratory procedures.

Section II. Analysis and Manipulation Technologies on a Chip

7. Microfluidic Drops as Microreactors

Charles N. Baroud

The use of individual droplets to transport reagents in microchannels solves three of the fundamental difficulties encountered in continuous flow microfluidic reactors: (i) The reagents are trapped inside the droplets, limiting their dispersion, (ii) mixing of species is performed by using the flow field inside the drop, and (iii) the ability to individually manipulate the drops allows for precise manipulation of small volumes of reagents. These advantages come at the price of increased complexity of the flows and of some new fluid mechanical issues. In this chapter, we begin by describing the advantages and by raising these fundamental issues, namely the determination of the shape, velocity, and transport properties within the drops. At the same time, ways of reducing the complexity associated with the fluid mechanics and the physical-chemistry are discussed. Later, we consider methods for producing and manipulating drops in microchannels with the aim of providing ways to perform reactions inside the drops. Two approaches to manipulation are discussed: passive manipulation, which relies on the channel geometry, and active manipulation which involves external forcing through electrical, mechanical, or optical methods. Drop manipulation is based on certain fundamental steps, namely the production, division, merging, or storing of drops, as well as the mixing of their contents. The implementation of these steps by passive methods is shown, which offers robust and simple control over many operations by taking advantage of careful design of the the microchannel geometry. This is followed by a discussion of active control methods which allow the dynamic tuning of the operations performed by passive means, as well as providing more complex operations. For instance, operations such as drop sorting are shown, as well as the synchronization of two drop formations or switching the order of two drops. These operations are only possible by introducing active control into the channels. Finally, example implementations on cellular and biochemical systems are discussed, as well as a discussion of future trends.

8. Optical Sectioning for Microfluidics

Yeh-Chan Ahn and Zhongping Chen

This chapter describes Doppler optical coherence tomography (OCT), a new optical tomographic technique that can image and quantify microstructure and flow simultaneously in microfluidic channel. Doppler OCT is a three-dimensional, non-contact, high-resolution, real-time imaging technique that provides information of wall location and shape in microchannel, three-dimensional velocity profile, and mixing performance. It is a versatile and essential tool for engineers and scientists who want to study transport phenomena in microchannel, to design and test microfluidic components, and to monitor a flaw or malfunction in lab-on-a-chip in situ. System configuration and principle of Doppler OCT are described and several applications are demonstrated.

9. Acquisition of Single Cell Data in an Optical Microscope

Kristin Sott, Emma Eriksson and Mattias Goksör

Data acquired on a single cell level has become increasingly important in the understanding of cellular behaviour. Traditionally, research in life science has focused on studies using ensemble averaging techniques. Since most cell models are based on averaged results from populations of cells, information regarding any heterogenic behaviour within the population is lost. Recent studies at the single cell level have revealed surprisingly heterogeneous behaviour, and highlight the importance of gaining detailed single cell data to update the models on cellular dynamics. We here present an experimental platform for analysing protein dynamics in single cells with high spatial and temporal control. The experimental platform utilizes the combination of the imaging resolution of a fluorescence microscope, with the spatial and temporal control of single cells and its environment provided by optical manipulation and microfluidics, respectively. The experimental platform is evaluated on the high osmolarity glycerol (HOG) signalling pathway in single Saccharomyces cerevisiae during environmental stress.

10. Elaborating Lab-on-a-Chips for Single-cell Transcriptome Analysis

Nathalie Bontoux, Luce Dauphinot and Marie-Claude Potier

Working at the single cell level is becoming incontrovertible in many fields of biology particularly when studying gene expression in complex tissues such as the brain. Gene expression protocols always start with the conversion of RNA to complementary DNA (cDNA) by reverse transcription. This low efficiency reaction is crucial since unconverted RNAs will not be analyzed further. In this chapter, a detailed protocol for single cell whole transcriptome analysis is presented. This protocol includes a novel microfluidics step for high yield reverse transcription performed in devices made of polydimethylsiloxane (PDMS). These devices allow the manipulation of nanoliter volumes, thus increasing the concentration of starting RNAs. This methodwas validated by comparing it to conventional protocols performed in microliter volumes using single cell amount of mouse brain RNA (10 pg). Single gene PCR was then integrated to the reverse transcription reaction on the same PDMS device in a separate chamber. The template switching PCR reaction for whole transcriptome amplification was, however, performed in conventional tubes since the yield was very poor in microfluidics devices because of molecular crowding. Gene profiling of single neuronal progenitors is discussed at the end of the chapter. Using this microfluidic approach (cell capture, lysis and reverse transcription in the microfluidic device followed by template switching PCR amplification in tube), a mean of 5000 genes were detected in each neuron, which corresponds to the expected number of genes expressed in a single cell. This demonstrates the outstanding sensitivity of the microfluidic method that was developed.

11. Integrated Circuit/Microfluidic Chips for Dielectric Manipulation

Thomas P. Hunt, D. Issadore, K.A. Brown, Hakho Lee and R.M. Westervelt

In this chapter, we describe the development of Integrated-Circuit/Microfluidic chips that can move individual living cells and chemical droplets along programmable paths using dielectrophoresis (DEP). These hybrid chips combine the biocompatibility of a microfluidic system with the complexity and programmability of an integrated circuit (IC), a microfluidic chamber is built directly on top of the IC and they offer new opportunities for sensing, actuation, and control. IC/Microfluidic chips can independently control the location of hundreds of dielectric objects, such as biological cells or chemical droplets, in the microfluidic chamber at the same time. The IC couples with suspended objects by using spatially patterned, time-dependent electromagnetic fields. The IC layout is similar to a computer display: it consists of a two-dimensional array of 128x256 metal 'pixels', each 11x11 μm² in size, controlled by a built-in SRAM memory. Each pixel can be energized by a radio frequency (RF) voltage up to 5 V_pp. The ICs were made in a commercial foundry, and a microfluidic chamber was built on its top surface at Harvard. Using this IC/Microfluidic chip, we have moved yeast and mammalian cells along programmed paths at speeds up to 300 μm/sec. Hundreds of cells can be individually trapped and simultaneously positioned into controlled patterns. The chip can trap and move pL droplets of water in oil, split one droplet into two, and mix two droplets into one, allowing one to conduct experiments with chemicals and individual cells, using tiny amounts of fluid. Our IC/Microfluidic chip provides a programmable platform that can individually control the motion of large numbers of cells and fluid droplets simultaneously for lab-on-a-chip applications.

12. Microchip-based PCR Amplification Systems

Nathaniel C. Cady

The polymerase chain reaction (PCR) has been widely used for amplification of DNA sequences. This technique can be coupled with a variety of detection assays for the identification of a wide range of DNA targets. Reducing PCR to the microchip level is of interest for portable detection technologies and high-throughput, massively parallel analytical systems. This chapter describes the basic process of creating a miniaturized, microfluidic PCR chip and a simple method for thermally cycling this microchip during PCR amplification. These methods can be broadly applied to a variety of microchip architectures, materials, and downstream analytical methods.

13. Cell Viability Measurement Using a Portable Photodiode Array Chip

Joon Myong Song and Ho Taik Kwo

Photodiode array (PDA) on-chip cell viability measurement is performed directly on the surface of PDA microchip. Cancer cells are treated with anticancer drugs such as naringenin (Nar), camptothecin (CAM) and sodium salicylate (Na-sal). The resultant apoptotic cells are then stained with trypan blue and spotted onto the surface of PDA microchip. This type of spotting eliminates the need of using complicated optical alignment, which is usually required for spectroscopic detection. The PDA microchip is used as a photodetector as well as a sample platform. PDA on-chip assay is based on the absorption detection of spotted cells. Light emitting diodes (LEDs) installed right above the PDA microchip produces red beam which reaches PDA microchip. Once the absorber is spotted on the surface of PDA microchip, the intensity of red beam which reaches PDA reduces as the amount of absorber increases. Trypan blue has a spectroscopic property to absorb the red beam. As the amount of trypan blue-stained cells increases, the PDA sense reduced intensity of red beam. Quantification of cell viability was accomplished using a calibration curve of red beam intensity versus the number of trypan blue-stained cells. The PDA on-chip cell viability measurement enables high-throughput measurement of optimal concentrations of different drugs against different cell lines in vitro.

14. A Charge-coupled Device (CCD) Based Optical Detector for Lab-on-a-Chip

Keith Herold and Avraham Rasooly

One important element of any Lab-on-a-chip is a detector that translates the physical assay into a convenient form. Among the many types of biodetection approaches (e.g. optical, electrochemical, piezoelectric and various others) optical detection is widely used. Here we describe a simple and relatively inexpensive charge-coupled device (CCD) camera based detector for monitoring chemiluminesce, florescence and colorimetric assays. The portable battery-operated detector includes an illumination source and a cooled CCD digital camera. The detector system, including camera operation, image acquisition and analysis, is controlled by a laptop computer. The level of detection of the system was found to be similar to the detection level of a commercial photomultipler based plate fluorometer. The multichannel CCD detector was used with an assay plate capable of testing nine samples simultaneously. The system is small and operated by a 12 volt source, so it is portable. The modular detector was designed to be used for a wide variety of optical detection modes including chemiluminesce, florescence and colorimetric assays and is suitable for point of care diagnostics and for providing healthcare to underserved population.

15. PCR Devices Using Glass Substrate

Hao Yu, Jianhua Qin and Bingcheng Lin

Polymerase chain reaction (PCR) provides an in vitro method for rapid enzymatic amplification of fragments of DNA and it is one of the important analytical tools in biological sciences that benefit from miniaturization. In the past few years, the possibility of performing fast and small volume PCR on a single device has attracted great interest and witnessed great advances. The miniaturization of PCR has been demonstrated to have distinct advantages including small sample requirement, short amplification time, rapid heating/cooling rates, and the potential to integrate other functional components to reduce power consumption and improve portability. In this chapter, the motivation of underlying miniaturized format of PCR will be firstly introduced. Moreover, the recent research and progress on chip substrates, chip design, surface modification, and the characteristics of two types of microchip PCR are summarized. Several issues related to PCR reaction volume, heating methods, amplification speed, and the product-detection methods are discussed. Furthermore, the example applications of these methods to perform miniaturized format of PCR on a glass substrate are demonstrated. In addition, the potential applications of microchip-PCR to integrate functional components such as pre-PCR and post-PCR, and other practical issues related to implementation of the miniaturized devices are discussed.

16. Braille Microfluidics

Tommaso F. Bersano-Begey, Yoko Kamotani and Shuichi Takayama

This chapter describes protocols for fabrication and assembly of microfluidic chips designed for Braille-display-based fluid actuation. Emphasis is placed on critical fabrication details such as channel cross-sectional shape and dimensions, and membrane thickness. Commercial sources for the Braille display hardware and some simple software operation are also described. A detailed example of a protocol for a chip used for immunoassays using the Braille display for fluid actuation is also described.

Braille Microfluidics is a microfluidic technology which uses arrays of small retractable pins on pre-existing Braille hardware as the actuation and control mechanism for elastomeric microfluidic chips. Braille actuation enables microfluidic pumping and valving without tubings and interconnects; the channels within the microfluidic chip are fabricated with a thin bottom membrane and are aligned over the Braille pins to interact with them. As the pins are raised or retracted they can perform all of the basic microfluidic operations such as valving, peristaltic pumping, and mixing.

Among the advantages of this setup are ease of fabrication (single layer and no interconnections), portability (the entire system can be configured to occupy just the size of a hand) and high-programmability (all pins, and hence all the microfluidic valves they actuate, can be independently addressed and controlled automatically through a computer with a USB connection). However the same setup also introduces new challenges, such as an increased evaporation rate through its thin bottom membrane and a pulsatile flow which is not always desirable. A few solutions to these problems along with applications in which the Braille system is used are briefly discussed.

17. Microfluidic Devices for Single-cell Analysis

Yan Chen and Jiang F. Zhong

Gene regulation is a continuous event. Differentiation/maturation of cells is orchestrated by sequentially expressing a series of genes within a cell after receiving signals from the microenvironment. Ultimately, the gene-gene interaction inside an individual cell determines the fate of that particular cell. Therefore, gene regulation should be studied at the single-cell level to understand how genes respond to environmental signals and subsequently produce proteins to regulate cellular activity. However, molecular biologists currently have limited ability to analyze the contents of a single-cell which typically are only several picoliters. Current devices and methods for biomedical research are designed for performing experiments at the microliter scale. Using those tools leads to significant material loss in single-cell analysis, and produces poor quality data.

To overcome the technical barrier of single-cell analysis, we have designed and constructed microfluidic devices with computer-controlled microvalves and peristaltic pumps for biochemical analysis at the nanoliter scale. Carrying out biochemical reactions at the nanoliter scale significantly reduces material loss from small volumes of analyte such as a single-cell. Equipped with a thermal stage, our devices can extract mRNA from 32 or more individual cells and convert mRNA to cDNA within 3 hours with 5 fold higher efficiency than bulk assays. The microfluidic devices we developed can analyze single-cell contents precisely and produce reliable data. Those devices have the potential to transform single-cell analysis from a challenge and expensive task to a routine assay for molecular biologists.